Chlamydia trachomatis is a bacterium with a unique

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1 Journal of Andrology, Vol. 32, No. 1, January/February 2011 Copyright E American Society of Andrology How to Detect Chlamydia trachomatis in Males? Review ADRIAN ELEY From the Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom. ABSTRACT: Much is known about the role of Chlamydia trachomatis in female infertility, although the same cannot be said about the organism s role in male infertility. Recently a number of researchers have provided a possible explanation of the pathogenesis of C trachomatis in male infertility and have suggested further studies. Unfortunately, current screening recommendations for C trachomatis in an infertile couple are vague and unhelpful, and many do not even mention this type of screening in the male. To enable any progress to be made in this field, it is essential that investigators know how best to detect C trachomatis, especially in the male. It is important, therefore, to know which specimen is best for C trachomatis detection, with respective strengths and weaknesses of each specimen. Similarly, it is equally important to have knowledge of which test is appropriate for the type of specimen being examined. First void urine is currently the specimen of choice for the routine detection of C trachomatis in males. Moreover, the best detection protocols in the developed world are based on molecular diagnosis of first void urine. These methods provide the best combination of sensitivity and specificity that is currently available on a clinical sample that can be self-taken. Interestingly, because semen is routinely collected for analysis in men of infertile couples, it has been suggested that protocols be developed for the optimal detection of C trachomatis in this specimen. Semen might provide additional information on infection of the upper genital tract, which may not be detected in first void urine. Finally, the importance of comparing tests for C trachomatis detection in updating our knowledge has been highlighted by the inability of some molecular methods to detect the new variant strain of C trachomatis. Key words: First void urine, semen, molecular diagnostics. J Androl 2011;32:15 22 Chlamydia trachomatis is a bacterium with a unique developmental cycle comprising infectious, metabolically inert elementary bodies (EBs) and noninfectious, metabolically active reticulate bodies. It is responsible for the most common sexually transmitted bacterial infection worldwide, affecting more than 90 million people (World Health Organization, 2001), and has been known for some time to have a significant impact on human reproduction (Paavonen and Eggert- Kruse, 1999), although its role in male infertility is still controversial (Ochsendorf, 2008). Nevertheless, our in vitro experiments have revealed that exposure of spermatozoa to chlamydial EBs can result in premature sperm death and stimulate an apoptosis-like response in sperm (Hosseinzadeh et al, 2001; Eley et al, 2005a,b). Interestingly, similar findings, which led to increased levels of sperm DNA fragmentation, were also found in in vivo studies (Satta et al, 2006; Gallegos et al, 2008), therefore providing a possible explanation of the pathogenesis of the organism in male infertility. Correspondence to: Dr Adrian Eley, Department of Infection and Immunity, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, United Kingdom ( a.r.eley@sheffield. ac.uk). Received for publication March 5, 2010; accepted for publication August 5, DOI: /jandrol There are different serovars or serotypes of the organism, with those commonly causing genital infections belonging to serovars D through K and with serovars D through G being the most common in the Western world (Donati et al, 2009). A new variant of C trachomatis serovar E (nvct) with a 377-bp deletion in the cryptic plasmid was recently detected in Sweden (Ripa and Nilsson, 2006, 2007). Interestingly, because some of the commercially available C trachomatis assays based on a plasmid target were unable to detect strains with the deletion in the plasmid, a large number of falsenegative results were discovered (Soderblom et al, 2006; Unemo et al, 2007). This resulted in the affected companies having to develop new molecular tests for C trachomatis diagnostics. Current screening recommendations for C trachomatis in an infertile couple are vague and unhelpful. In the United Kingdom, the Royal College of Obstetricians and Gynaecologists (1999) and the National Institute for Health and Clinical Excellence recommend that before undergoing uterine instrumentation, women (presumably in an attempt to prevent spread of infection) should be offered screening for C trachomatis using an appropriately sensitive technique. If the result of a test for C trachomatis is positive, women and their sexual partners should be referred for appropriate management with treatment and contact tracing. One 15

2 16 Journal of Andrology N January ÙFebruary 2011 Table. Strengths and weaknesses of different clinical specimens used for detecting Chlamydia trachomatis Specimen Strengths Weaknesses Urethral swab Quality of specimen can be assured Not easy to collect and painful Urine Easy to collect Cannot culture the organism Semen Routinely collected for semen analysis Inhibiting to cell culture and sometimes to NAATs Serum Relatively easy to collect Not from the infection site source (Institute for Clinical Systems Improvement) has described symptoms of a possible genital tract infection in the male, although no further information was provided to help investigate the cause. A number of sources have commented on the finding of significant numbers of leukocytes in a urethral smear, first void urine, or semen as indicative of a possible genital tract infection but gave no additional information as to how that should be investigated further (Male Infertility Best Practice Policy Committee of the American Urological Association, 2006; European Association of Urology). The only more specific advice is found in the European Association of Urology guidelines and is from an older reference of Taylor-Robinson (1997), who stated that the ideal diagnostic test for C trachomatis in semen has not yet been established despite modern DNA detection techniques. Interestingly, in spite of the above United Kingdom guidelines, Sowerby and Parsons (2004) found that 53% of in vitro fertilization clinics in the United Kingdom neither screen the woman nor give antibiotic prophylaxis, and only 4% screen the male partner. In the screening of sperm, egg, and embryo donors, recent guidance (Association of Biomedical Andrologists, 2008) has suggested that all donors be screened for C trachomatis before and after donation, according to the strategy developed by the British Association for Sexual Health and HIV (2006). However, it is not clear how best to detect C trachomatis in the male because many different methods have been employed over the years. The aim of this review is to assess established methods of detection in the light of newer technical developments. Which Specimen? Essentially, there are 4 possible specimens that may be examined, each with its respective strengths and weaknesses (Table). Historically, urethral swabs were collected as part of the genital examination within the genitourinary medicine clinic and were required for chlamydial culture. Unfortunately, in males, the taking of urethral swabs may be painful because it requires deep insertion, and trying to limit the procedural pain can have a negative effect on specimen quality. The idea of using self-taken, noninvasive specimens such as urine, especially when combined with antigen detection tests such as the enzyme immunoassay (EIA), has resulted in a move away from routine use of urethral swabs. Comparative studies have shown that although urethral swabs gave higher rates of detection than first void urine for both sensitivity and specificity (81.0% vs 76.2% and 99.0% vs 95.5%, respectively) when an EIA test was used (Sellors et al, 1991), the difference between the specimens was reduced when a nucleic acid amplification test (NAAT) was used (96.2% vs 92.5% and 100% vs 100%, respectively) (Carroll et al, 1998). A recent systematic review has confirmed the latter finding (Cook et al, 2005). Since the early 1990s, first void or first catch urine (usually the first ml) has been shown to be an acceptable specimen for the detection of C trachomatis genital infection in men (Chernesky et al, 1990). Apart from the previously described changes in methodology for improved C trachomatis detection in urine, another factor has emphasized the advantages of using urine. A recent study has shown that there is no significant difference in organism load between first void urine and urethral swabs in men when quantitative polymerase chain reaction (PCR) is used for assessment (Michel et al, 2007). However, it has been known for a while that NAAT inhibitors can be present in many clinical specimens, and there is some controversy on the extent of NAAT inhibition in urine (Mahony et al, 1998; Toye et al, 1998; Van der Pol et al, 2001). Therefore, detection of C trachomatis in urine is advantageous because the sample is self-taken, noninvasive, and contains a high organism load (although there can be NAAT inhibitors present). In routine diagnosis of chlamydial infection in the male, a semen specimen would not normally be requested, but semen specimens are routinely collected in infertile men to test semen quality. Moreover, such a specimen might provide additional information as to whether there is a chlamydial infection of the upper genital tract. Unfortunately, there is no approved methodology for testing of semen for C trachomatis (Chernesky, 2005; Peeling and Embree, 2005), and this poses a serious problem. The question as to whether semen is a suitable sample for detection of C trachomatis in infertile men has yet to be answered. The identification of chlamydial antibodies is clearly an indirect approach to detect C trachomatis in any

3 Eley N C trachomatis and Male Infertility 17 clinical specimen (usually serum but can be semen). Moreover, there is no single sensitive and specific C trachomatis antibody test that has been consistently used to investigate chlamydial serology (Johnson and Horner, 2008), and this deficiency makes it difficult to compare results from different studies. Among commercially available assays, there is often variable sensitivity and specificity because there may be crossreactivity with Chlamydophila (Chlamydia) pneumoniae (Gijsen et al, 2001), a common respiratory pathogen. It is obvious, therefore, that there are limitations with the use of chlamydial serology (in whatever test specimen) in general, to detect the presence of C trachomatis. Which Test? There are a number of laboratory methods to detect C trachomatis that range from cell culture to NAATs, as discussed next. Other research methods that are not generally available in a diagnostic laboratory, such as electron microscopy, will not be discussed. Culture In the early days of C trachomatis screening, the bacterium was grown on cell monolayers such as McCoy and inoculated with the clinical specimen such as a urethral swab (Mardh et al, 1980). The characteristic chlamydial inclusion body can be detected when stained with a fluorescent antibody that binds to either the chlamydial major outer membrane protein or lipopolysaccharide (LPS). Older nonspecific stains, including Giemsa or iodine, have lower sensitivities and are not recommended for routine use (Stamm et al, 1983). However, with the introduction of noninvasive samples such as urine, cell culture proved too insensitive (Taylor-Robinson and Thomas, 1991), with sensitivities of less than 70% compared with direct immunofluorescence (DIF) (Taylor-Robinson, 1997). Culture sensitivity can be enhanced by blind passaging, which increases the number of infected cells that can be detected by staining. It was also discovered that components of semen were toxic to the growth and maintenance of the monolayer, and a solution to this toxicity problem was the dilution of the semen to decrease the toxic effect; however, this decrease in toxicity was achieved at the expense of sensitivity to detect C trachomatis (Tjiam et al, 1987). Culture is now regarded as slow, labor intensive, and hence quite costly. Nevertheless, it has excellent specificity and allows for antibiotic susceptibility testing and serovar determination, which may be advantageous in epidemiologic studies. Antigen Detection There are 2 commercial approaches to antigen detection, and these are EIA and DIF. EIA tests are usually based on the ability to detect chlamydial LPS with an anti-lps antibody. The chlamydial LPS extract from the clinical sample is added to the anti-lps antibody, which coats wells of a microtiter plate. Upon binding of LPS to the antibody, a colorimetric reaction occurs that can be measured in a microplate reader. The test is easy to perform but only has a sensitivity of 10 5 to 10 7 organisms/ml. It also can be cross-reactive with other bacteria such as staphylococci, Bacteroides species, and Escherichia coli (Eggert- Kruse et al, 1995; Ivanov et al, 2009). The test specificity can be improved by retesting any positive reactions with a confirmatory blocking antibody or by using the DIF or direct fluorescence antibody test. The ability to automate these tests has led to a possibility of highthroughput testing. The DIF or direct fluorescence antibody test uses an antibody that is tagged with a fluorescent label and is able to detect chlamydial EBs directly from smears made from clinical specimens. Smears are then examined under a fluorescence microscope. Cross-reactions are not usually a problem because the morphology of EBs is distinctive. It is the only method that allows for confirmation of specimen quality. The sensitivity of the method is higher than that of the EIA test (and can be increased further by low-speed centrifugation of specimens before staining) (Taylor-Robinson and Thomas, 1991) and is related to the minimum number of EBs that are counted (which can be in the range of 1 10) and the proficiency of the person reading the smear. Unfortunately, a skilled and experienced microscopist is required for optimal performance. DIF has a sensitivity of 80% to 90% and specificity of 98% to 99% relative to the culture method when both are performed optimally (Black, 1997). Molecular Testing These tests that detect either DNA or RNA can be divided into those that amplify or do not amplify the target. A test not amplifying the target typically uses a probe to detect ribosomal RNA (Gen-Probe PACE 2 assay) and has a sensitivity of approximately 10 4 organisms/ml. Although it has a similar sensitivity level as culture, this test has the advantage of good specimen stability characteristics, whereas culture requires strict transport and handling criteria. However, amplified molecular tests such as PCR show much greater sensitivity with a theoretic single nucleic acid sequence that can be detected. A systematic review in 2002 established that both PCR and ligase chain reaction (LCR) showed better sensitivities than nonmolecular methods (Watson et al, 2002). The authors also concluded that these NAATs used with noninvasive samples such as urine were more effective at detecting asymptomatic chlamydial infection and that these tests performed well in low-prevalence populations. Skidmore et al (2006) documented the 2 NAATs currently in widespread use in the United Kingdom: the

4 18 Journal of Andrology N January ÙFebruary 2011 Becton Dickinson ProbeTec, which uses strand displacement amplification (SDA) technology (Spears et al, 1997), and the Roche Cobas Amplicor PCR (which is now being superseded by a real-time PCR method, the Roche Cobas TaqMan CT). A third NAAT currently available in the United Kingdom is transcriptionmediated amplification by Gen-Probe (Pasternack et al, 1997). Such methods are widely available in the developed world, including Europe and the United States. It is now generally accepted that in C trachomatis detection, NAATs have become the method of choice (Hamdad and Orfila, 2005; Gaydos et al, 2008) so much so that they must be used in the United Kingdom National Chlamydia Screening Programme (United Kingdom Department of Health). The first NAAT to be used successfully for the detection of C trachomatis in males was PCR using inhouse methodology. This test has been used subsequently in a number of studies involving men of infertile couples (Ochsendorf et al, 1999; Hosseinzadeh et al, 2004; Gdoura et al, 2008). Since then, PCR has been developed commercially so that the method is standardized (eg, the Roche Cobas Amplicor PCR has an built-in control for specimen inhibitors [as does LCR, as described below]) (Hamdad-Daoudi et al, 2004; de Barbeyrac et al, 2006). A more recent development has been the introduction of real-time or quantitative PCR, which allows detection of C trachomatis DNA copy numbers (Al-Mously et al, 2009). In the 1990s, a commercial system for LCR (Abbott), similar to Amplicor, also proved to be successful at detecting C trachomatis in men of infertile couples (Eggert-Kruse et al, 1997; Fujisawa et al, 1999; Eggert-Kruse et al, 2002a,b). However, the LCR commercial system was discontinued several years ago and is no longer available. Serology Until recently, there was no consensus about the detection of chlamydial IgG and IgA in serum and the presence of C trachomatis in the male genital tract. It was generally believed that detection of serum IgG and IgA was of no diagnostic value in male infertility (Wolff et al, 1994; Dieterle et al, 1995). Studies by Radouani et al (1996), Weidner et al (1996), and Levy et al (1999) confirmed a lack of correlation between serologic results and direct detection of C trachomatis in semen. However, recent studies (Idahl et al, 2004, 2007) have shown that the presence of C trachomatis IgG and IgA antibodies in serum from the male partner of an infertile couple was correlated with pregnancy (as confirmed by routine ultrasound at gestation weeks 15 17) and associated with subtle negative changes in semen characteristics. In particular, C trachomatis serum IgA in men correlated with reduced chances of achieving pregnancy (P 5.021; relative risk [RR] ; 95% confidence intervals [CI], ), and the chance was further reduced in combination with C trachomatis IgG (P 5.001; RR ; 95% CI, ) (Idahl et al, 2007). The role of chlamydial IgG and IgA antibodies in semen and their relation to semen quality is perhaps more controversial. Many studies have shown no association (Eggert-Kruse et al, 1997, 1998; Habermann and Krause, 1999; Penna Videau et al, 2001; Liu and Zhu, 2003), whereas others studies have shown an association (Wolff et al, 1991; Ochsendorf et al, 1999). A recent study also showed no relationship between past or present C trachomatis infection, defined by positive direct and serologic markers (in serum and/or semen), and quality of semen defined according to World Health Organization parameters such as sperm count, motility, and morphology (de Barbeyrac et al, 2006). Perhaps the fundamental problem is that the presence of chlamydial IgG or IgA antibodies in serum or semen does not allow a distinction between past or present genital infection (Dieterle et al, 1995). Therefore, an appropriate current summary suggests that the determination of C trachomatis antibodies in serum or seminal plasma seems to be of limited diagnostic value in male infertility work-up (de Barbeyrac et al, 2006). Comparisons Between the Specimens and Tests Not all of the 4 specimens discussed previously are currently suitable and/or recommended for testing. Urethral swabs are probably the best samples to examine and are required for culture. However, for the preceding reasons, first void urine samples are now more commonly examined, although urethral swabs are still an alternative specimen. Semen is rarely tested, and there are no recommended methods to test it. Serum and/or semen antibodies both provide limited information and are controversial. Therefore, for the purpose of this review, our comparisons of different tests will be limited to the examination of urine. Although urine is probably not quite as good a specimen as a urethral swab in terms of sensitivity, testing a centrifuged deposit of urine has been shown to be as sensitive as testing a urethral swab if the same test is applied to each (Thomas et al, 1991). Nevertheless, testing of urine in cell cultures is too insensitive, with a positivity rate of only 23.7% in men (Chernesky et al, 1990). Although EIA is easy to perform, sensitivity levels can be low, varying between 62% and 97% (Taylor-Robinson and Thomas, 1991). Specificity can also be an issue. In studies of men of infertile couples, prevalence rates using EIA were high 25.19% and 33.33%, respectively (Bornman et al, 1998; Mania-

5 Eley N C trachomatis and Male Infertility 19 Pramanik et al, 2001). DIF shows improved sensitivity when compared with EIA and has value in looking at nonviable chlamydiae in specimens, owing to prolonged transport and suboptimal storage; however, the technique requires an experienced microscopist. Therefore, amplified testing of urine is considered the most effective way for diagnosing infections in men, and the effectiveness has been shown when making comparisons with Gen-Probe PACE 2 and EIA (Chernesky et al, 2003). Although specificity was high for both tests, Gen-Probe PACE 2 only had a sensitivity of 65.9% in comparison with LCR, which had a sensitivity of 90.1% (Carroll et al, 1998). When LCR was applied to testing of men of infertile couples, detection rates were low at 0% and 4.54%, respectively (Eggert-Kruse et al, 1997; Bollmann et al, 2001). An advantage of LCR was that it was only available commercially so that studies using this test were comparable. However, many studies with PCR have used in-house methodologies that are not comparable. A recent example of such methodology applied to men of infertile couples in Tunisia gave a prevalence rate of 39.4% (Gdoura et al, 2008). Amplicor, which is only available commercially, is a PCR-based method. When this test was applied to men of infertile couples, low prevalence rates of 5.4% and 0%, respectively, were found (Hamdad-Daoudi et al, 2004; Rosemond et al, 2006). More recently, ProbeTec SDA has been tested in the same population group, and a prevalence rate of 3.52% was found (Kokab et al, 2010). A relatively early comparison of amplified methods then available (Amplicor, SDA, and transcriptionmediated amplification) showed that they performed equally well (Templeton et al, 2001). Since then, a number of new real-time methods, including Roche COBAS TaqMan CT (Hadad et al, 2009) and Abbott real-time CT assay (Walsh et al, 2009), have come on the market. Comparative studies of new real-time methods and existing NAATs have been made but with limitations. For example, Abbott real-time CT compared with ProbeTec SDA showed increased sensitivity (Walsh et al, 2009). In a more comprehensive study, comparisons were made for Abbott real-time CT, Roche COBAS TaqMan CT, and Gen-Probe tests (Aptima Combo 2 and Aptima C trachomatis assay, which target 23S rrna and 16S rrna, respectively). Although patient numbers were small, the Abbott real-time CT showed better sensitivity (59/83 vs 57/83) (Moller et al, 2008). However, both tests underperformed in comparison with the Gen-Probe tests because the latter tests were able to detect the new variant C trachomatis strain (Magbanua et al, 2007), whereas the other tests could not. Conclusions and Future Directions For the routine detection of C trachomatis in males, first void or first catch urine is currently the specimen of choice because these methods provide the best combination of sensitivity and specificity that are currently available on a clinical sample that can be self-taken. Nevertheless, urethral swabs are a useful, alternative specimen and are essential if culture of the organism is required. Semen might provide additional information on detection, but there are still no approved methods for detecting C trachomatis in semen. Therefore, until comparative studies are performed with, for example, first void urine, no recommendations can be made. Current serologic methods cannot be recommended mostly on grounds of poor specificity. Unless culture is required, the best detection protocols in the developed world are based upon molecular diagnosis of first void urine as discussed earlier, although some may not be able to detect the new variant strain. In countries that do not have access to molecular techniques, the best test would be to use EIA on first void urine. However, choice of specimen is often influenced by a number of local factors, including the population size of the group to be screened, prevalence rate in the population, predictive values of the tests being used, and whether testing is performed in a laboratory. All of these factors will contribute to the final decision. Because semen is routinely collected for analysis in men of infertile couples, it is suggested that protocols be developed for the optimal detection of Ctrachomatisin this specimen. There are suggestions that semen might provide additional information on infections of the upper genital tract that may not be detected in first void urine (Eggert-Kruse et al, 1997; Bornman et al, 1998; Fujisawa et al, 1999; Rosemond et al, 2006; Gdoura et al, 2008; Gallegos-Avila et al, 2009; Kokab et al, 2010; Mazzoli et al, 2010). Moreover, recent recommendations for future testing of semen for Ctrachomatishave been proposed (Eley and Pacey, 2010). They include making a comparison of commercially available NAATs as well as comparing tests of semen with testing of first void urine, the use of an internal control for inhibitors, and confirmation of any positive results using the minimum testing algorithm. Finally, the importance of comparing tests for C trachomatis detection in updating our knowledge has been highlighted by the inability of some molecular methods to detect the new variant strain. References Al-Mously N, Cross NA, Eley A, Pacey AA. Real-time polymerase chain reaction shows that density centrifugation does not always

6 20 Journal of Andrology N January ÙFebruary 2011 remove Chlamydia trachomatis from human sperm. Fertil Steril. 2009;92: Association of Biomedical Andrologists, Association of Clinical Embryologists, British Andrology Society, British Fertility Society, Royal College of Obstetricians and Gynaecologists. UK guidelines for the medical and laboratory screening of sperm, egg and embryo donors. Hum Fertil (Camb). 2008;11: Black CM. Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin Microbiol Rev. 1997;10: Bollmann R, Engel S, Petzoldt R, Gobel UB. Chlamydia trachomatis in andrologic patients direct and indirect detection. Infection. 2001;29: Bornman MS, Ramuthaga TN, Mahomed MF, Greef AS, Crewe- Brown HH, Reif S. Chlamydial infection in asymptomatic infertile men attending an andrology clinic. Arch Androl. 1998;41: British Association for Sexual Health and HIV. Sexually transmitted infections: UK national screening and testing guidelines, August Available at: Accessed March 3, Carroll KC, Aldeen WE, Morrison M, Anderson R, Lee D, Mottice S. Evaluation of the Abbott LCx ligase chain reaction assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and genital swab specimens from a sexually transmitted disease clinic population. J Clin Microbiol. 1998;36: Chernesky M, Castriciano S, Sellors J, Stewart I, Cunningham I, Landis S, Seidelman W, Grant L, Devlin C, Mahony J. Detection of Chlamydia trachomatis antigens in urine as an alternative to swabs and cultures. J Infect Dis. 1990;161: Chernesky M, Jang D, Chong S, Sellors J, Mahony J. Impact of urine collection order on the ability of assays to identify Chlamydia trachomatis infections in men. Sex Transm Dis. 2003;30: Chernesky MA. The laboratory diagnosis of Chlamydia trachomatis infections. Can J Infect Dis Med Microbiol. 2005;16: Cook RL, Hutchison SL, Ostergaard L, Braithwaite S, Ness RB. Systematic review: noninvasive testing for Chlamydia trachomatis and Neisseria gonorrhoeae. Ann Intern Med. 2005;142: de Barbeyrac B, Papaxanthos-Roche A, Mathieu C, Germain C, Brun JL, Gachet M, Mayer G, Bebear C, Chene G, Hocke C. Chlamydia trachomatis in subfertile couples undergoing an in vitro fertilization program: a prospective study. Eur J Obstet Gynecol Reprod Biol. 2006;129: Dieterle S, Mahony JB, Luinstra KE, Stibbe W. Chlamydial immunoglobulin IgG and IgA antibodies in serum and semen are not associated with the presence of Chlamydia trachomatis DNA or rrna in semen from male partners of infertile couples. Hum Reprod. 1995;10: Donati M, Di Francesco A, D Antuono A, Pignanelli S, Shurdhi A, Moroni A, Baldelli R, Cevenini R. Chlamydia trachomatis serovar distribution and other concurrent sexually transmitted infections in heterosexual men with urethritis in Italy. Eur J Clin Microbiol Infect Dis. 2009;28: Eggert-Kruse W, Neuer A, Clussmann C, Boit R, Geissler W, Rohr G, Strowitzki T. Seminal antibodies to human 60kd heat shock protein (HSP 60) in male partners of subfertile couples. Hum Reprod. 2002a;17: Eggert-Kruse W, Rohr G, Demirakca T, Rusu R, Naher H, Petzoldt D, Runnebaum B. Chlamydial serology in 1303 asymptomatic subfertile couples. Hum Reprod. 1997;12: Eggert-Kruse W, Rohr G, Probst S, Rusu R, Hund M, Demirakca T, Aufenanger J, Runnebaum B, Petzoldt D. Antisperm antibodies and microorganisms in genital secretions a clinically significant relationship? Andrologia. 1998;30(suppl 1): Eggert-Kruse W, Rohr G, Strock W, Pohl S, Schwalbach B, Runnebaum B. Anaerobes in ejaculates of subfertile men. Hum Reprod Update. 1995;1: Eggert-Kruse W, Zwick EM, Batschulat K, Rohr G, Armbruster FP, Petzoldt D, Strowitzki T. Are zinc levels in seminal plasma associated with seminal leukocytes and other determinants of semen quality? Fertil Steril. 2002b;77: Eley A, Hosseinzadeh S, Hakimi H, Geary I, Pacey AA. Apoptosis of ejaculated human sperm is induced by co-incubation with Chlamydia trachomatis lipopolysaccharide. Hum Reprod. 2005a; 20: Eley A, Pacey AA. The value of testing semen for Chlamydia trachomatis in males of infertile couples. Int J Androl. In press. Epub ahead of print 19 Aug Eley A, Pacey AA, Galdiero M, Galdiero M, Galdiero F. Can Chlamydia trachomatis directly damage your sperm? Lancet Infect Dis. 2005b;5: European Association of Urology. Dohle GR, Diemer T, Giwercman A, Jungwirth A, Kopa Z, Krausz C. Guidelines on male infertility. Available at: pdf. Accessed May 28, Fujisawa M, Nakano Y, Matsui T, Okada H, Arakawa S, Kamidono S. Chlamydia trachomatis detected by ligase chain reaction in the semen of asymptomatic patients without pyospermia or pyuria. Arch Androl. 1999;42: Gallegos G, Ramos B, Santiso R, Goyanes V, Gosalvez J, Fernandez JL. Sperm DNA fragmentation in infertile men with genitourinary infection by Chlamydia trachomatis and Mycoplasma. Fertil Steril. 2008;90: Gallegos-Avila G, Ortega-Martinez M, Ramos-Gonzalez B, Tijerina- Menchaca R, Ancer-Rodriguez J, Jaramillo-Rangel G. Ultrastructural findings in semen samples of infertile men infected with Chlamydia trachomatis and mycoplasmas. Fertil Steril. 2009;91: Gaydos CA, Ferrero DV, Papp J. Laboratory aspects of screening men for Chlamydia trachomatis in the new millennium. Sex Transm Dis. 2008;35:S45 S50. Gdoura R, Kchaou W, Ammar-Keskes L, Chakroun N, Sellemi A, Znazen A, Rebai T, Hammami A. Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples. J Androl. 2008;29: Gijsen AP, Land JA, Goossens VJ, Leffers P, Bruggeman CA, Evers JL. Chlamydia pneumoniae and screening for tubal factor subfertility. Hum Reprod. 2001;16: Habermann B, Krause W. Altered sperm function or sperm antibodies are not associated with chlamydial antibodies in infertile men with leucocytospermia. J Eur Acad Dermatol Venereol. 1999;12: Hadad R, Fredlund H, Unemo M. Evaluation of the new COBAS TaqMan CT test v2.0 and impact on the proportion of new variant Chlamydia trachomatis by the introduction of diagnostics detecting new variant C trachomatis in Orebro County, Sweden. Sex Transm Infect. 2009;85: Hamdad F, Orfila J. Diagnosis of urogenital infection by Chlamydia trachomatis. Contribution of genetic amplification techniques. Prog Urol. 2005;15: Hamdad-Daoudi F, Petit J, Eb F. Assessment of Chlamydia trachomatis infection in asymptomatic male partners of infertile couples. J Med Microbiol. 2004;53: Hosseinzadeh S, Brewis IA, Eley A, Pacey AA. Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death. Hum Reprod. 2001;16:

7 Eley N C trachomatis and Male Infertility 21 Hosseinzadeh S, Eley A, Pacey AA. Semen quality of men with asymptomatic chlamydial infection. J Androl. 2004;25: Idahl A, Abramsson L, Kumlin U, Liljeqvist JA, Olofsson JI. Male serum Chlamydia trachomatis IgA and IgG, but not heat shock protein 60 IgG, correlates with negatively affected semen characteristics and lower pregnancy rates in the infertile couple. Int J Androl. 2007;30: Idahl A, Boman J, Kumlin U, Olofsson JI. Demonstration of Chlamydia trachomatis IgG antibodies in the male partner of the infertile couple is correlated with a reduced likelihood of achieving pregnancy. Hum Reprod. 2004;19: Institute for Clinical Systems Improvement. The diagnosis and management of basic infertility. Available at: diagnosis_and_management_of_infertility_2301.html. Accessed March 3, Ivanov IB, Kuzmin MD, Gritsenko VA. Microflora of the seminal fluid of healthy men and men suffering from chronic prostatitis syndrome. Int J Androl. 2009;32: Johnson AM, Horner P. A new role for Chlamydia trachomatis serology. Sex Transm Infect. 2008;84: Kokab A, Akhondi MM, Sadeghi MR, Modarresi MH, Aarabi M, Jennings R, Pacey AA, Eley A. Raised inflammatory markers in semen from men with asymptomatic chlamydial infection. J Androl. 2010;31: Levy R, Layani-Milon MP, Giscard D Estaing S, Najioullah F, Lornage J, Aymard M, Lina B. Screening for Chlamydia trachomatis and Ureaplasma urealyticum infection in semen from asymptomatic male partners of infertile couples prior to in vitro fertilization. Int J Androl. 1999;22: Liu DB, Zhu PY. Detection of IgG and IgM antibodies against Chlamydia trachomatis in semen of asymptomatic infertile patients. Zhonghua Nan Ke Xue. 2003;9: Magbanua JP, Goh BT, Michel CE, Aguirre-Andreasen A, Alexander S, Ushiro-Lumb I, Ison C, Lee H. Chlamydia trachomatis variant not detected by plasmid based nucleic acid amplification tests: molecular characterisation and failure of single dose azithromycin. Sex Transm Infect. 2007;83: Mahony J, Chong S, Jang D, Luinstra K, Faught M, Dalby D, Sellors J, Chernesky M. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcriptionmediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol. 1998;36: Male Infertility Best Practice Policy Committee of the American Urological Association; Practice Committee of the American Society for Reproductive Medicine. Report on optimal evaluation of the infertile male. Fertil Steril. 2006;86(5 suppl 1):S202 S209. Mania-Pramanik J, Gokral J, Meherji PK. Chlamydia trachomatis infection among asymptomatic males in an infertility clinic. Indian J Dermatol Venereol Leprol. 2001;67: Mardh P-A, Colleen S, Sylwan J. Inhibitory effect on the formation of chlamydial inclusions in McCoy cells by seminal fluid and some of its components. Invest Urol. 1980;17: Mazzoli S, Cai T, Addonisio P, Bechi A, Mondaini N, Bartoletti R. Chlamydia trachomatis infection is related to poor semen quality in young prostatitis patients. Eur Urol. 2010;57: Michel CE, Sonnex C, Carne CA, White JA, Magbanua JP, Nadala EC Jr, Lee HH. Chlamydia trachomatis load at matched anatomic sites: implications for screening strategies. J Clin Microbiol. 2007;45: Moller JK, Pedersen LN, Persson K. Comparison of Gen-Probe transcription-mediated amplification, Abbott PCR, and Roche PCR assays for detection of wild-type and mutant plasmid strains of Chlamydia trachomatis in Sweden. J Clin Microbiol. 2008;46: National Institute for Health and Clinical Excellence, National Collaborating Centre for Women s and Children s Health. Fertility: assessment and treatment for people with fertility problems. Available at: cg011fullguideline.pdf. Accessed March 3, Ochsendorf FR. Sexually transmitted infections: impact on male fertility. Andrologia. 2008;40: Ochsendorf FR, Ozdemir K, Rabenau H, Fenner T, Oremek R, Milbradt R, Doerr HW. Chlamydia trachomatis and male infertility: Chlamydia-IgA antibodies in seminal plasma are C trachomatis specific and associated with an inflammatory response. J Eur Acad Dermatol Venereol. 1999;12: Paavonen J, Eggert-Kruse W. Chlamydia trachomatis: impact on human reproduction. Hum Reprod Update. 1999;5: Pasternack R, Vuorinen P, Miettinen A. Evaluation of the Gen-Probe Chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women. J Clin Microbiol. 1997;35: Peeling R, Embree J. Screening for sexually transmitted infection pathogens in semen samples. Can J Infect Dis Med Microbiol. 2005;16: Penna Videau S, Cermeno Vivas J, Salazar N. IgA antibodies to Chlamydia trachomatis and seminal parameters in asymptomatic infertile males. Arch Androl. 2001;46: Radouani F, Bennani A, Takourt B, Hda N, Ibrahimi S, Boutaleb Y, Guinet R, Benslimane A. Chlamydial infections and male infertility in Morocco. Contracept Fertil Sex. 1996;24: Ripa T, Nilsson P. A variant of Chlamydia trachomatis with deletion in cryptic plasmid: implications for use of PCR diagnostic tests. Euro Surveill. 2006;11:E Ripa T, Nilsson PA. A Chlamydia trachomatis strain with a 377-bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests. Sex Transm Dis. 2007;34: Rosemond A, Lanotte P, Watt S, Sauget AS, Guerif F, Royere D, Goudeau A, Mereghetti L. Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples. Pathol Biol (Paris). 2006;54: Royal College of Obstetricians and Gynaecologists Evidence-Based Clinical Guidelines. Guideline Summary No. 2: the initial investigation and management of the infertile couple. Br J Urol Int. 1999;83: Satta A, Stivala A, Garozzo A, Morello A, Perdichizzi A, Vicari E, Salmeri M, Calogero AE. Experimental Chlamydia trachomatis infection causes apoptosis in human sperm. Hum Reprod. 2006;21: Sellors J, Mahony J, Jang D, Pickard L, Castriciano S, Landis S, Stewart I, Seidelman W, Cunningham I, Chernesky M. Rapid, onsite diagnosis of chlamydial urethritis in men by detection of antigens in urethral swabs and urine. J Clin Microbiol. 1991;29: Skidmore S, Horner P, Mallinson H. Testing specimens for Chlamydia trachomatis. Sex Transm Infect. 2006;82: Soderblom T, Blaxhult A, Fredlund H, Herrmann B. Impact of a genetic variant of Chlamydia trachomatis on national detection rates in Sweden. Euro Surveill. 2006;11:E Sowerby E, Parsons J. Prevention of iatrogenic pelvic infections during in vitro fertilization current practice in the UK. Hum Fertil (Camb). 2004;7: Spears PA, Linn CP, Woodard DL, Walker GT. Simultaneous strand displacement amplification and fluorescence polarization detection of Chlamydia trachomatis DNA. Anal Biochem. 1997;247:

8 22 Journal of Andrology N January ÙFebruary 2011 Stamm WE, Tam M, Koester M, Cles L. Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluoresceinconjugated monoclonal antibodies. J Clin Microbiol. 1983;17: Taylor-Robinson D. Evaluation and comparison of tests to diagnose Chlamydia trachomatis genital infections. Hum Reprod. 1997; 12(suppl 11): Taylor-Robinson D, Thomas BJ. Laboratory techniques for the diagnosis of chlamydial infections. Genitourin Med. 1991;67: Templeton K, Roberts J, Jeffries D, Forster G, Aitken C. The detection of Chlamydia trachomatis by DNA amplification methods in urine samples from men with urethritis. Int J STD AIDS. 2001;12: Thomas B, Gilchrist C, Taylor-Robinson D. Detection of Chlamydia trachomatis by direct immunofluorescence improved by centrifugation of specimens. Eur J Clin Microbiol Infect Dis. 1991;10: Tjiam KH, van Heijst BY, Polak-Vogelzang AA, Rothbarth PH, van Joost T, Stolz E, Michel MF. Sexually communicable microorganisms in human semen samples to be used for artificial insemination by donor. Genitourin Med. 1987;63: Toye B, Woods W, Bobrowska M, Ramotar K. Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis testing. J Clin Microbiol. 1998;36: Unemo M, Olcen P, Agne-Stadling I, Feldt A, Jurstrand M, Herrmann B, Persson K, Nilsson P, Ripa T, Fredlund H. Experiences with the new genetic variant of Chlamydia trachomatis in Orebro county, Sweden proportion, characteristics and effective diagnostic solution in an emergent situation. Euro Surveill. 2007; 12:E5 6. United Kingdom Department of Health. Chlamydia screening programme rollout core requirements. Available at: PublicationsPolicyAndGuidance/DH_ Accessed March 3, Van der Pol B, Ferrero DV, Buck-Barrington L, Hook E 3rd, Lenderman C, Quinn T, Gaydos CA, Lovchik J, Schachter J, Moncada J, Hall G, Tuohy MJ, Jones RB. Multicenter evaluation of the BDProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol. 2001;39: Walsh A, Rourke FO, Laoi BN, Crowley B. Evaluation of the Abbott realtime CT assay with the BD ProbeTec ET assay for the detection of Chlamydia trachomatis in a clinical microbiology laboratory. Diagn Microbiol Infect Dis. 2009;64: Watson EJ, Templeton A, Russell I, Paavonen J, Mardh P-A, Stary A, Pedersen BS. The accuracy and efficacy of screening tests for Chlamydia trachomatis: a systematic review. J Med Microbiol. 2002;51: Weidner W, Floren E, Zimmermann O, Thiele D, Ludwig M. Chlamydial antibodies in semen: search for silent chlamydial infections in asymptomatic andrological patients. Infection. 1996; 24: Wolff H, Neubert U, Volkenandt M, Zochling N, Schlupen E-M, Bezold G, Meurer M. Detection of Chlamydia trachomatis in semen by antibody-enzyme immunoassay compared with polymerase reaction, antigen-enzyme immunoassay, and urethral cell culture. Fertil Steril. 1994;62: Wolff H, Neubert U, Zebhauser M, Bezold G, Korting HC, Meurer M. Chlamydia trachomatis induces an inflammatory response in the male genital tract and is associated with altered semen quality. Fertil Steril. 1991;55: World Health Organization. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections Overviews and Estimates. Geneva, Switzerland: World Health Organization; 2001.